The goal of the research is to understand the mechanism of muscle thin filament regulation at the molecular level. To activate contractile and motile events, Ca2+ binds to a transducer in order to transmit information about the intracellular Ca2+ concentration via interaction with target proteins. In striated muscles, Ca2+ binds to troponin C (TnC). TnC in complex with TnI and TnT cooperatively activates the actin-containing thin filament through tropomyosin (TM)bound along the length of the filament. Molecular genetics and protein design will be used to address fundamental questions about TnC function and the conserved N-terminus of TM. The x-ray structure of TnC has two globular Ca2+ binding domains connected by an extended central helix. Aim 1 is to test the hypothesis that the flexibility of the central helix is important for function, as proposed for calmodulin (CaM). The central helix will be replaced with different structures: alpha-helix, flexible random coil, or a rigid polyproline II helix. Though the structures of TnC and CaM are closely related, TnC has a N- terminal alpha-helix that CaM lacks. Aim 2 is to define the function of the N-helix in TnC, its role in activating the thin filament in the presence of Ca2+, and its importance in stabilizing TnC. In Aims 1 and 2, mutants will be made using oligonucleotide-directed mutagenesis, mutant proteins will be expressed in E. coli, and purified proteins will be studied using established assays for function and conformation. Though there are widely used in vitro and in situ assays for TnC function, there is no assay in living cells. Aim 3 is to develop a system to analyze TnC function in cultured mouse muscle C2 cells. TnC expression will be inhibited using antisense oligonucleotides. Cells will be transfected with vectors encoding wildtype or mutant TnCs. The consequences on myofibril assembly and contractility will be analyzed. The conserved N-terminus of striated alpha-TM is crucial for actin binding and regulatory function. Aim 4 describes experiments to use N-terminal coiled coil synthetic peptides to determine the structure N-terminus (NMR and x-ray crystallography), folding mechanism (circular dichroism, calorimetry, NMR and role of N-terminal acetylation in stabilizing the coiled coil.